Reference Database for the 18S Rrna Gene

Home , Amphidomataceae, Azadinium spinosum, Blastodinium, Histioneis, Protista taxonomy, Suessiales

Received: 3 November 2017 | Revised: 15 February 2018 | Accepted: 24 February 2018 DOI: 10.1111/1755-0998.12781

RESOURCE ARTICLE

DINOREF: A curated dinoflagellate (Dinophyceae) reference database for the 18S rRNA gene

1Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Naples, Italy Abstract 2Sorbonne Universite, CNRS, UMR Dinoflagellates are a heterogeneous group of protists present in all aquatic ecosys- Adaptation et Diversite en Milieu Marin, tems where they occupy various ecological niches. They play a major role as primary Station Biologique, Roscoff, France producers, but many species are mixotrophic or heterotrophic. Environmental Correspondence metabarcoding based on high-throughput sequencing is increasingly applied to Diana Sarno, Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Naples, assess diversity and abundance of planktonic organisms, and reference databases Italy. are definitely needed to taxonomically assign the huge number of sequences. We Email: [email protected] provide an updated 18S rRNA reference database of dinoflagellates: DINOREF. Funding information Sequences were downloaded from GENBANK and filtered based on stringent quality Italian Ministry of Education, University and Research criteria. All sequences were taxonomically curated, classified taking into account classical morphotaxonomic studies and molecular phylogenies, and linked to a series

of metadata. DINOREF includes 1,671 sequences representing 149 genera and 422 species. The taxonomic assignation of 468 sequences was revised. The largest number of sequences belongs to Gonyaulacales and Suessiales that include toxic and

symbiotic species. DINOREF provides an opportunity to test the level of taxonomic resolution of different 18S barcode markers based on a large number of sequences and species. As an example, when only the V4 region is considered, 374 of the 422

species included in DINOREF can still be unambiguously identified. Clustering the V4 sequences at 98% similarity, a threshold that is commonly applied in metabarcoding studies, resulted in a considerable underestimation of species diversity.

KEYWORDS 18S rRNA gene, dinoflagellates, diversity, phylogeny, sequence database, V4 region

1 | INTRODUCTION precise ways to enumerate them. DNA-based detection and enumera- tion methodologies, such as high-throughput sequencing (HTS) Assessing global biodiversity constitutes an important and urgent task metabarcoding of environmental samples, now offer opportunities for in the face of the currently unprecedented rate of climate change, but assessing protistan diversity rapidly and precisely (Amaral-Zettler, this task is fraught with major challenges. A large part of this biodiver- McCliment, Ducklow, & Huse, 2009; Massana et al., 2015; Piredda sity is composed of protists, and it is especially in these unicellular et al., 2017; Stoeck et al., 2009; de Vargas et al., 2015). Yet, to trans- eukaryotes that taxonomists are confronted by the fact that cell mor- late these HTS data into species occurrences requires a comprehen- phology does not always allow discrimination of species, especially in sive reference database. Curated databases of reference sequences small and relatively featureless taxa. When compared with morpho- linked to taxonomically identified specimens constitute important logical traits, sequence data usually provide more precise and appar- research infrastructures for the advancement of our knowledge of the ently more objective ways to delineate species and therefore more protistan diversity (Decelle et al., 2015; Morard et al., 2015).

Dinoflagellates form a large phylum of protists distributed in vari- dinoflagellate classification at the suprageneric level, we also present ous aquatic ecosystems (Hackett, Anderson, Erdner, & Bhattacharya, an alternative organization of the sequence data, namely one based 2004; Not et al., 2012). The lineage includes autotrophs, hetero- on results of 18S phylogenetic inferences integrated with the latest trophs and mixotrophs; most species are free-living, but parasites literature sources. Finally, we illustrated the degree to which the 18S and symbionts are abundant as well, adding complexity to aquatic V4 region, which is broadly used in metabarcoding surveys and has ecological networks (Gomez, 2012b; Jephcott et al., 2016; Stoecker, been proposed as the universal eukaryotic prebarcode (Hu et al., 1999; Weisse et al., 2016). Dinoflagellates are of socio-economic rel- 2015; Pawlowski et al., 2012), is able to discriminate species. evance because several species produce secondary metabolites, some of which are highly toxic to humans and marine organisms 2 | MATERIALS AND METHODS (Anderson, Cembella, & Hallegraeff, 2012; Berdalet et al., 2016). Therefore, dinoflagellates are monitored on a regular basis in many 2.1 | Sequence retrieval coastal regions. The assessment of dinoflagellate diversity is still largely based on light microscopy observations (LM). Yet, this approach Dinoflagellate 18S rRNA entries available on the 29th of August 2016 has its limitations: it requires a high level of taxonomic expertise and were downloaded from NCBI GENBANK (https://www.ncbi.nlm.nih.gov/) is time-consuming, and many species, including minute, naked and using the following text query: Dinophyceae[Organism] AND (small parasitic dinoflagellates, are virtually impossible to identify. In some subunit ribosomal[titl] OR 18S[titl] OR SSU[titl]). The features associ- cases, toxic species are difficult to distinguish from nontoxic relatives ated with the GENBANK entries were extracted. Sequences of early in LM (de Salas, Laza-Martınez, & Hallegraeff, 2008; Montresor, branching dinoflagellates, not classified as Dinophyceae (sensu Orr John, Beran, & Medlin, 2004). et al. (2012)), were recovered genus by genus and were excluded from

Despite its rather low variability (Murray, Flø Jørgensen, Ho, Patter- DINOREF, but provided as supplementary material (see below). son, & Jermiin, 2005), the 18S ribosomal subunit (18S) is potentially a good DNA barcode region for dinoflagellates because of the large number of 2.2 | Sequence verification sequences deposited in public repositories as a result of its popular use in taxonomic and phylogenetic studies (Gomez, 2014; John et al., 2014), as Sequences labelled as “environmental samples” in GENBANK (i.e., from well as in single-cell PCR amplification studies (e.g., Gomez, Moreira, & metabarcoding, clone libraries, uncultured organisms—labelled as

Lopez-Garc ıa, 2010; Hoppenrath, Murray, Sparmann, & Leander, 2012; Ki, ENV in the GENBANK locus field) and those classified above the genus

Jang, & Han, 2005; Ruiz Sebastian & O’Ryan, 2001). The variable V4 or V9 level were removed. Retained sequences were aligned with MAFFT regions in the 18S rRNA-encoding region are the most commonly used version 7 (default parameters, Katoh & Standley, 2013) and manually nucleotide markers in metabarcoding studies on environmental samples checked. Sequences not meeting the following quality criteria were (e.g., Le Bescot et al., 2016; Onda et al., 2017). removed (Figure 1, step 1): (i) sequences deposited as 18S but not To improve taxonomic annotation of environmental sequences representing 18S at all or only the very 30-end of it; and (ii) 18S generated by HTS approaches, curated reference barcode databases sequences not including the full V4 region. Of the remaining 2 are needed. PR was the first database that became available for pro- sequences, the regions outside the 18S rRNA gene were removed. tists (Guillou et al., 2013), which included 136,866 nuclear-encoded After a second filtration step (Figure 1, step 2), only those sequences sequences that were taxonomically assigned to eight taxonomic that fulfilled all of the following criteria were retained: (i) sequence fields. Few other specialized databases have been created to provide length ≥450 bp; (ii) <50 ambiguous nucleotides; (iii) <20 consecutive taxonomically validated sequences with updated nomenclature and ambiguous bp; (iv) insertion <20 consecutive bp; and v. deletions contextual metadata for different groups of organisms (e.g., Decelle <100 consecutive bp. Sequences that aligned poorly or not at all et al., 2015 for 16S of photosynthetic eukaryotes; Morard et al., with any of the other sequences were blasted against GENBANK and

2015 for foraminifera). were removed if BLAST results suggested placement outside dinoflag-

The aim of this study was to provide a taxonomically curated ellates (i.e., BLAST assignation <90%). The retained set of sequences database, called DINOREF, composed of the “core dinoflagellates,” that constituted the “quality-checked database” (Figure 1). is, the species with a dinokaryon, a modified nucleus containing per- manently condensed fibrillar chromosomes, as defined by Orr, Mur- 2.3 | Taxonomic validation ray, Stuken,€ Rhodes, and Jakobsen (2012). To populate the database, we gathered all dinoflagellate 18S rRNA sequences available in The taxonomic validation was an iterative process including the

GENBANK, screened them against a set of quality criteria and verified following steps: their taxonomic assignations by means of phylogenetic positioning.

For each sequence, taxonomy and nomenclature from GENBANK were 1. control on the validity of the nomenclature, based on Fensome updated, deploying a phylogenetic approach, and taking into account et al. (1993) and Gomez (2012a); ALGAEBASE (Guiry & Guiry, 2017; the most recent literature and taxonomy databases. We organized http://www.algaebase.org), CEDIT (Hoppenrath & Elbr€achter, 2015; the 18S sequences into the same suprageneric ranks as those used http://www.dinophyta.org) and information from the literature of 2 in PR (Guillou et al., 2013). Given the “dynamic status” of the specific groups (Figure 1, step 3); MORDRET ET AL. | 3

FIGURE 1 Workflow describing the steps applied to generate the curated and annotated dinoflagellate 18S rRNA Reference Database DINOREF from the sequences downloaded from NCBI

2. phylogenetic evidences based on the primary tree, the genus- A number of long sequences (≥1,700 bp), representing the diversity level trees and the taxonomic reference tree (Figure 1, step 4). within each genus, were selected to build a reduced taxonomic reference tree (Figure 1, step 4). In case the only reference for a given genus Species names were validated following taxonomically accepted was represented by a shorter sequence, its phylogenetic placement was names in ALGAEBASE (Guiry & Guiry, 2017; names marked as “C”). verified building a “rough tree” based on a shorter alignment, but these Sequences included in the quality-checked database were aligned with shorter sequences were excluded from the alignment used to build the

MAFFT version 7, and a phylogenetic tree was built using FASTTREE (Price, reduced taxonomic reference tree. The data set was aligned using MAFFT

Dehal, & Arkin, 2010) as implemented in the GENEIOUS software (Kearse and the tree built with RAXML (Stamatakis, 2014). Branch support was et al., 2012). This primary tree provided information on the number, statisti- established using 100 bootstrap replicates. This reduced tree provided cal support and position of the different terminal clades (Figure 1, step 4). a clearer representation of dinoflagellate phylogeny and enabled checks In order to identify sequences attributed to the wrong genus in on the phylogenetic relationships for the terminal clades, allowing the

GENBANK, we aligned the sequences labelled with the same genus name removal of any remaining nondinoflagellate sequence from the quality- using MAFFT and visualized them in Seaview (Gouy et al., 2010). When checked database (Figure 1, step 5). This set of taxonomically curated possible (three or more sequences per genus), a maximum-likelihood sequences was called the “taxonomically checked database.” tree was inferred using PHYML version 3.0 (Guindon et al., 2010). Branch support was established using 100 bootstrap replicates. Gen- 2.4 | Database finalization era represented by less than three sequences were grouped with their closest phylogenetic groups after verifying their positioning in the pri- From the taxonomically checked database, a final nonredundant mary tree. Outlying sequences in the generic trees were sought in the sequence alignment (i.e., only containing unique sequences) was pro- primary tree, and if they were found inside the clade of another genus, duced using MAFFT and a majority-rule consensus tree (RAXML, 100 boot- they were renamed accordingly. straps) was built (Figure 1, step 6). Every sequence in the taxonomically 4 | MORDRET ET AL. checked database was labelled using a standardized eight term-ranked 3 | RESULTS taxonomy, that is, Kingdom, Supergroup, Division, Class, Order, Family, 2 Genus and Species in the same way as in PR (Guillou et al., 2013). The 3.1 | The DINOREF database database includes also an additional classification of the sequences into different “Superclades” defined according to the results of phylogenetic 3.1.1 | Phase 1: Sequence verification inferences and specialized literature (Figure 1, step 7).

The standard metadata extracted from GENBANK (see Supplemen- A total of 6,175 GENBANK sequences were downloaded with the given tary Material 1 for a complete list) has been supplemented by infor- search criteria. Upon removal of sequences not assigned at a genus mation on: (i) species habitat (Guiry & Guiry, 2017; Hoppenrath, or species level, 4,199 were retained, and of these, 2,223 remained Murray, Chomerat, & Horiguchi, 2014), (ii) potential toxicity following removal of non-18S sequences as well as 18S sequences (Moestrup et al., 2009) and (iii) symbiotic or parasitic lifestyle derived in which the V4 region was incomplete or lacking. The verification from original papers (Figure 1, step 8). The taxonomically checked process resulted in a database of 1,684 aligned, good-quality database integrated with metadata constitutes “DINOREF (dinoflagel- sequences. late reference database)”. 3.1.2 | Phase 2: Taxonomic validation 2.5 | V4 analysis The primary tree inferred from the quality-checked database To compare the resolution power of the V4 barcode region with that revealed that genera constituted the best-supported taxonomic level; of the full-length 18S gene, the V4 fragments were extracted from the that is, they were usually monophyletic with high bootstrap support alignment using the V4 primers described in Piredda et al. (2017). (data not shown). Three genera were found to be represented by Sequences of the V4 region were then dereplicated and split by genus more than 150 sequences each: Alexandrium with 210 sequences, and Superclade using Mothur (Schloss et al., 2009). Each group of Gambierdiscus with 169 sequences and Symbiodinium with 173 sequences was then re-aligned using MAFFT and checked manually. For sequences (around 36% of the sequences in the database; Table 2). each Superclade and each genus, distance matrices of pairwise differ- Some species within these three genera were represented by a large ences between sequences over the length of the V4 (p-distance) were number of different 18S sequences. For instance, 54 slightly differ- generated using the software MEGA version 7 (Kumar, Stecher, & ent sequences were attributed to Gambierdiscus scabrosus. The num- Tamura, 2016). Boxplots of distances were produced with R (R Devel- ber of species included in other genera was much lower: a first opment Core Team, 2016) using the “ggplot2” library (Wickham, group of 27 genera included each between 68 and 10 sequences 2009). V4 OTUs at 98% similarity were generated clustering the (about 40% of the total number of sequences), a second group of 55 sequences with VSEARCH algorithm with “distance-based greedy cluster- genera contained between nine and three sequences (18% of the ing” (DGC, Rognes, Flouri, Nichols, Quince, & Mahe, 2016) as imple- total), whereas the remaining 63 genera were represented by one or mented in Mothur. The V4 unique sequences and the V4 OTUs at two sequences only (6% of the total) (Figure 4). 98% similarity are provided as supplementary materials (see below). In The resulting taxonomically checked database contained 1,671 those files, we also listed the 18S sequences sharing the same V4 dinoflagellate sequences, corresponding to 1,540 unique sequences region and the V4 sequences clustering in the same OTU at 98%. and belonging to 149 genera (Table 1) and 422 species (Table 2). 2 DINOREF has been incorporated into PR version 4.9.0 which is Thirteen of the original 1,684 sequences had to be removed because available at https://doi.org/10.6084/m9.figshare.5913181. they did not belong to dinoflagellates. The assignation of 468

Supplementary data that include the DINOREF database as an Excel sequences (28% of the total database) had to be revised because the file (Supplementary Material 1), a full-length 18S sequences fasta names originally assigned to them were synonyms, invalid or the phy- text file (Supplementary Material 2) associated with three different logenetic analyses revealed that they were attributed to the wrong taxonomy files, (i) the original taxonomy of GENBANK entry (Supple- taxon. Sequence length ranged from 579 to 1,764 bp (Figure S1), mentary Material 3), (ii) the curated taxonomy (Supplementary Mate- with 1,200 of them being full or nearly full length (between 1,600 rial 4) and (iii) the curated taxonomy including the Superclade and 1,764 bp). The majority of sequences between 1,100 and classification used in this study (Supplementary Material 5), are avail- 1,400 bp originated from single-cell amplifications. able in flat file format on Figshare (https://doi.org/10.6084/m9. figshare.5568454). The format of the fasta and taxonomy text files 3.1.3 | Phase 3: Database finalization is compatible with Mothur (Schloss et al., 2009) and Qiime (Capo- raso et al., 2010) tools. Fasta and taxonomy files can be opened with The curated sequences were organized hierarchically, following the 2 a text editor. We also provide two Excel files with V4 sequences 8-level taxonomic framework used in the PR database (Columns E-L and V4 OTUs at 98% similarity (Supplementary Materials 6 and 7). in Supplementary Material 1). For the assignation of the “order” Supplementary Material 8 includes a fasta file containing all level, we followed a conservative approach accepting the following sequences of early branching dinoflagellates recovered from GENBANK six orders: Gonyaulacales, Peridiniales, Dinophysiales, Prorocentrales, but not included in DINOREF. Suessiales and Gymnodiniales (“Order” in Table 1; column I in MORDRET ET AL. | 5

Supplementary Material 1). Some sequences could not be placed There was an incomplete and unbalanced 18S sequence represen- within any of these orders and were listed as Dinophyceae ordo tation of described taxa, and the number of available sequences dif- incertae sedis. Assignment at the family level was problematic due fered considerably among Superclades, clades and genera (Table 2). to the still unresolved phylogenetic relationship among genera (see For example, the order Suessiales included 14 genera represented by Section 4). We generally followed the classification provided by 18S sequences and 11 without reference 18S sequence, while

ALGAEBASE (Guiry & Guiry, 2017). Gonyaulacales were reasonably well covered (17 genera with sequences and three without). Overall, only 422 of the 2,342 (18%) taxonomically recognized dinoflagellate species were found to have a 3.2 | Superclades: an attempt to depict the current reference 18S sequence (Guiry & Guiry, 2017) (Table 2). When consid- organization of dinoflagellates ering genera, only 149 of the 232 genera had a reference sequence.

A majority-rule consensus tree built with the 1,540 unique The DINOREF database includes also a broad set of metadata sequences provided a phylogenetic representation of the 18S retrieved from GENBANK and/or from the taxonomic literature (Supple- sequences contained in the DINOREF database. In this tree, a large mentary Material 1). Most sequences (89%) originated from marine number of clades with ≥50% bootstrap support collapsed onto a ecosystems, 8% from freshwater and 2% from brackish, estuarine or polytomy (Figure 2). The well-supported clades of the majority-rule continental saline habitats. Specific lifestyle information (i.e., sym- consensus tree (Table 1; Figure 2) were grouped by us into higher biont or parasite and their host) is available for 302 sequences taxonomic ranks (“Superclade” in Table 1; Column D in Supplemen- (18%). In addition, 24% of the sequences were annotated as benthic, tary Material 1) reflecting the current taxonomic organization based and 34% as belonging to potentially toxic species. on previously published phylogenies and multigene phylogenies (see selected references in Table 1). 3.3 | The barcoding V4 region The largest Superclade included species of the order Gonyaula- cales (Superclade 1) and was recovered from the tree in five well- If only the V4 region is considered, the number of unique DINOREF supported clades. Within it, clade 1A (Table 1; Figure 2) encom- sequences shrunk from 1,540 to 946 (Supplementary Material 6). passed a large number of sequences of the genera Alexandrium and This decrease in sequence number was mostly due to slightly differ- Gambierdiscus, while the other contained either a single genus (clade ent intraspecific 18S sequences sharing identical V4 ribotypes, that 1D, Gonyaulax) or different but phylogenetically closely related gen- is, haplotypes, unique sequences of ribosomal genes. Nonetheless, a era (e.g., clade 1B, Ceratium and Tripos). large proportion of the intraspecific diversity, observed when the Sequences of the order Dinophysiales (Superclade 2) grouped whole 18S was taken into account, was also detectable when only into three clades, while Prorocentrales (Superclade 11) were dis- the V4 marker was considered. For example, the single morphologi- tributed over five distinct clades. cally defined species Alexandrium fundyense and Gambierdiscus Sequences attributed to the order Peridiniales sensu lato were scabrosus were represented by 103 and 54 18S and 36 and 39 V4 considerably diversified in the 18S phylogeny, and seven Superclades sequences, respectively. However, cases occurred in which different were identified: Thoracosphaeraceae (7 clades), Peridiniales sensu species shared the same V4 ribotype; For example, V4 failed to dis- stricto (4 clades), Kryptoperidiniaceae, Heterocapsaceae and tinguish toxic Azadinium spinosum from nontoxic A. trinitatum, Kare- Podolampadaceae, each with one clade; one Superclade only nia brevis from K. mikimotoi and seven Dinophysis species from one included a pair of genera (Ensiculifera and Pentapharsodinium), and another (Supplementary Material 6). Moreover, there are cases in only one included a single genus (Blastodinium). which sequences belonging to closely related genera shared identical All sequences of the order Suessiales (Superclade 3) grouped in a V4 regions; as is the case of the V4 #788 shared among Karlodinium single clade and sequences of species classified within Gymno- veneficum, Takayama pulchellum and Takayama acrotrocha, and the diniales sensu lato clustered into six distinct Superclades (Super- V4 #315 which is identical in several Histioneis spp. and Ornithocer- clades 12–17), of which Superclade 13 included all Gymnodiniales cus spp. (Supplementary Material 6). Overall, 374 of the 422 species sensu stricto. Two Superclades of Gymnodiniales sensu lato corre- included in DINOREF could be identified with the V4 barcode marker. sponded to Torodiniales and Kareniaceae, respectively, and the other In general, Superclades with many sequences deriving from sev- three Superclades contained sequences attributed to a single genus, eral genera showed large pairwise p-distances for the V4 region (Fig- which are Amphidinium, Akashiwo and Gyrodinium. ure 3). Yet patterns differed profoundly among the Superclades. For Two Superclades, that is, Tovelliaceae and Ptychodiscales, were example, Superclade 1 (Gonyaulacales) that contained 543 sequences classified as “Dinophyceae ordo incertae sedis” in ALGAEBASE (Guiry & from 17 genera and 92 species showed similar p-distance to the Guiry, 2017). A series of dinoflagellate sequences, often including smaller Superclade 8 (Peridiniales sensu stricto) that had only 116 taxa of uncertain classification, were resolved in a whole series of sequences from 13 genera and 46 species (Table 2; Figure 3). Other small clades, and even single sequences, all emerging from the poly- Superclades, such as #16 (Amphidinium) or #18 (Tovelliaceae) and tomy. We distributed these sequences into two categories: “Uncer- #19 (Blastodinium), showed high levels of variation even though tain naked dinophyceae (UND)” and “Uncertain thecate dinophyceae there were only a small number of sequences (Table 2; Figure 3). (UTD)”. Superclade 2 (Dinophysiales) showed similar p-distance patterns as 6 | MORDRET ET AL.

TABLE 1 Organization of the dinoflagellate 18S sequences into genera, clades and Superclades

(Continued) MORDRET ET AL. | 7

TABLE 1 (Continued)

Clades represent the statistically supported (bootstrap values ≥50) larger clades of the majority-rule consensus tree. Clades are grouped into Superclades based on recent taxonomic literature; a selection of references supporting the identification of Superclades is reported. Dinoflagellate species included in DINOREF, but lacking morphological or phylogenetic evidence to be placed within any Superclade has been grouped in “Uncertain Thecate Dinophyceae” and “Uncertain Naked Dinophyceae.” Names in bold represent genera included in the multigene dinoflagellate phylogeny by Orr et al. (2012). Colours identifying clades, and Superclades correspond to those used in the tree (Figure 2).

#3 (Suessiales), but included less than half as many sequences and a the same genus and 12 OTUs included sequences from different far lower number of genera and species. Within genera, the largest genera. Remarkably, a single OTU (OTU #126 in Supplementary p-distance values were found for Amphidinium, Coolia, Gambierdiscus, material 7) contained sequences from 59 genera belonging to differ- Gonyaulax, Protoperidinium and Togula (Figure 4). ent Superclades (e.g., Karlodinium, Prorocentrum, Gyrodinium, When clustered into OTUs at 98% similarity, the 946 unique V4 Podolampas, Duboscquodinium). On the other hand, sequences sequences were reduced to 313 OTUs (Supplementary Material 6); belonging to the same species clustered in different OTUs (e.g., 33 of these OTUs included sequences from different species within Alexandrium fundyense Group_I and Gambierdiscus scabrosus). 8 | MORDRET ET AL.

TABLE 2 Number of unique and total dinoflagellate 18S rRNA gene sequences by Superclade included in the database

No. of sequences in No. of taxa in Total no. of DINOREF DINOREF described taxa

Superclades Unique Total Genera Species Genera Species # 1 Gonyaulacales 507 543 17 85 20 296 # 2 Dinophysiales 97 97 8 38 13 358 # 3 Suessiales 223 240 14 29 26 91 # 4 Thoracosphaeraceae 71 82 16 22 19 66 # 5 Amphidomataceae 26 27 2 11 2 20 # 6 Kryptoperidiniaceae 21 23 5 10 6 16 #7 Pentapharsodinium-Ensiculifera 662 4 26 # 8 Peridiniales sensu stricto 106 116 13 46 25 475 # 9 Heterocapasaceae 18 20 1 7 1 16 # 10 Podolampadaceae 7 7 4 7 8 42 # 11 Prorocentrales 70 78 2 28 4 68 #12 Akashiwo 8131 1 1 1 # 13 Gymnodiniales sensu stricto 115 129 16 41 21 341 # 14 Kareniaceae 31 38 4 9 8 40 #15 Gyrodinium 15 15 1 7 3 112 #16 Amphidinium 36 40 1 10 3 101 # 17 Torodiniales 9 9 2 3 2 3 # 18 Tovelliaceae 6 10 4 4 4 19 #19 Blastodinium 29 32 1 8 1 13 # 20 Ptychodiscales 1 1 1 1 1 2 UTD Uncertain Thecate Dinophyceae 56 56 23 32 37 172 UND Uncertain Naked Dinophyceae 82 89 11 19 25 84 Total 1,540 1,671 149 422 232 2,342

Number of dinoflagellate genera and species represented in the database by at least one sequence. Sequences not assigned to the species level (annotated as “sp.”) were not considered. Total number of genera and species described (based on Gomez (2012a), ALGAEBASE (Guiry & Guiry, 2017), CEDIT (Hoppenrath & Elbr€achter, 2015).

4 | DISCUSSION defined taxa in multiple clades emerging directly from a polytomy neither supports nor falsifies the natural status of these taxa; mor- 2 DINOREF adds to the already available 18S protists databases PR (Guil- phological evidence is simply not backed up by molecular evidence. lou et al., 2013) and SILVA (Quast et al., 2013) a considerable number Whether this polytomy is hard, that is, real, due to a brief period of of new 18S sequences, provides an updated and validated identifica- rapid radiation, or soft, due to paucity of data, remains to be tion for more than 400 entries, and places each sequence within a resolved. One promising approach is multigene phylogenies that start curated 8-level taxonomic framework. This is a “conservative” hierar- to provide a clearer picture of dinoflagellate evolutionary patterns chical system, largely reflecting the one presented in ALGAEBASE (Guiry (Janouskovec et al., 2017; Orr et al., 2012). Some groups, such as & Guiry, 2017). In addition, we also present a tentative classification Gymnodiniales, appear to be polyphyletic and are in need of taxo- of dinoflagellates based on the 18S phylogeny and supported by nomic revision. As a matter of fact, various papers have been recent taxonomic literature (“Superclade” in Table 1; column D in recently published to clarify the taxonomic position of several groups

Supplementary Material 1). DINOREF also includes sequences of the (e.g., Boutrup, Moestrup, Tillmann, & Daugbjerg, 2016; Yuasa, Hori- V4 region with information on this marker’s capability to discriminate guchi, Mayama, & Takahashi, 2016). It is clear that the taxonomic among species. treatment of the genera needs revision. For example, genera such as The marked polytomy of the 18S tree built with the sequences Protoperidinium are paraphyletic with daughter genera inside them included in DINOREF leaves the phylogenetic status of most of the (Gu, Liu, & Mertens, 2015; Liu et al., 2015; Mertens et al., 2015), higher taxa unresolved. These results are in line with what was suggesting that these paraphyletic genera need to be split. known from previous studies carried out with this ribosomal marker The comparison between the number of dinoflagellate species

(e.g., Bachvaroff et al., 2014; Murray et al., 2005; Saldarriaga, Taylor, estimated by recent checklists (Gomez, 2012a) and ALGAEBASE (Guiry Keeling, & Cavalier-Smith, 2001). Recovery of morphologically & Guiry, 2017) and those for which sequences are available in MORDRET ET AL. | 9

FIGURE 2 Consensus phylogenetic tree (RAXML, GTR model) based on 1,540 unique 18S rRNA sequences in the DINOREF. Alignment of 2,153 bp with three sequences of Ciliates (U97109; X56165 and X03772) and three sequences of Apicomplexa (M97703; AF236097 and AF291427) used as outgroup. Clades are ordered according to their size and are supported by bootstrap values ≥50%; black dots are proportional to bootstrap values. The colours of the Superclades and clades correspond to those in Table 1. Clades within each Superclade have been marked (A, B, C, etc.), along the outer rim of the tree, corresponding to their assignment in this figure. The Superclades “Uncertain Naked Dinophyceae” and “Uncertain Thecate Dinophyceae” have not been marked and neither have the small clades on the upper left of the tree. The tree can be visualized on ITOL version 3—Interactive Tree of Life (Letunic and Bork, 2016, at https://itol.embl.de/tree/ 1932052318357911479398328) in which all clades are marked

FIGURE 3 (a) Barplot showing the number of genera with 18S rRNA information in 19 of the 20 Superclades depicted in Table 1 and Figure 2. Superclade 20 (Ptychodiscales) is not shown as it contains only one sequence. The number of sequences in each Superclade is reported on the top of each bar. (b) Boxplot showing the pairwise p-distances of the V4 region in the 19 dinoflagellate Superclades 10 | MORDRET ET AL.

FIGURE 4 Boxplots showing the range of the pairwise p-distances over the V4 region of the 18S rRNA sequences within the genera included in each Superclade (indicated by number and colour code as in Table 1 and Figure 2). Number of sequences that have been used to calculate pairwise p-distance and number of species represented by those sequences are specified for each genus. Sequences with no species name (annotated “sp.”) were not accounted for in the number of species, but still used for the calculation of pairwise p-distance MORDRET ET AL. | 11

DINOREF shows that all the taxonomic groups (defined here as Super- Phylogenetic studies and metabarcoding approaches will provide clades) have representatives in the 18S data set, but not all of them important information in this direction in the coming years. We have are equally well covered. The coverage of the DINOREF database is shown the good resolution capability of the V4 18S rDNA marker for strongly biased towards sequences of species in the focus of global dinoflagellates, but other barcode regions are worth exploring such as research such as toxic species (e.g., Alexandrium, Gambierdiscus) and conserved regions of the LSU (e.g., Grzebyk et al., 2017). We recom- endosymbionts associated with coral reefs (Symbiodinium). In general, mend testing different potential markers in parallel with the production it is also biased towards autotrophs because these organisms are of new 18S reference sequences, because a larger amount of data is more easily grown in culture and therefore have been characterized required to achieve the best possible solution for dinoflagellates and more easily than heterotrophs and mixotrophs, which are difficult to protists in general. isolate and may require alternative approaches such as single-cell sequencing. The DINOREF database is still far from covering dinoflagellate diversity. Only 18% of the 2,342 described species according to NOTE ADDED IN PROOF ALGAEBASE (Guiry & Guiry, 2017) are represented by an 18S reference Proposed taxonomic revisions are being incorporated in NCBI GEN- sequence. The 422 dinoflagellate species with an 18S sequence pre- BANK. sent in DINOREF represent a marked increase from the around 150 species reported by Murray et al. (2005) and from the 345 by Gomez (2014) showing that sequence information is increasing ACKNOWLEDGEMENTS rapidly as a result of the description of new species from different SM has been supported by a PhD fellowship from Stazione Zoo- geographic areas. However, there are still 83 genera and 1,639 spe- logica Anton Dohrn (SZN). This study was supported by the pro- cies for which no 18S sequences are available yet. ject FIRB Biodiversitalia (RBAP10A2T4) funded by the Italian In some cases, however, the diversity of species defined based on Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR). We morphological characters is probably overestimated. This may be the are grateful to Dr. Chris Bowkett for improving the use of English case of species attributed to the genus Gymnodinium, where 38% of in the manuscript. the 270 described species have not been reported since their original description (Thessen, Patterson, & Murray, 2012). However, there are also genera such as Symbiodinium and Scrippsiella in which a marked AUTHOR CONTRIBUTION intrageneric diversity has been uncovered by molecular approaches S.M., D.S., R.P., W.H.C.F.K., D.V. and M.M. designed the research. S.M. (Gottschling et al., 2012; Pochon, Putnam, & Gates, 2014). and R.P. collected data and the designed framework for molecular and The usefulness of metabarcoding using the V4 region of the 18S computational analyses. S.M. assembled the database and provided a rRNA gene depends on how well the region is able to distinguish the first draft of the manuscript; all authors contributed to its final version. various taxa of interest (Bendif et al., 2014; Hu et al., 2015). For dinoflagellates, it is considered variable enough to discern most of the species (Ki, 2012; Smith, Kohli, Murray, & Rhodes, 2017) although DATA ACCESSIBILITY there are some exceptions. Our results show that the V4 region can DINOREF is available in flat file format on Figshare at https://doi.org/ unambiguously discriminate 374 of the 422 species included in DI- 10.6084/m9.figshare.5568454 and is incorporated in the Protist NOREF. On the upside, this outcome illustrates the capability of the V4 2 barcode region to resolve protist diversity (Hu et al., 2015; Ki, 2012), Ribosomal Reference database (PR ) version 4.9.0 which is available but on the downside, the V4 is unable to discriminate between some at https://doi.org/10.6084/m9.figshare.5913181. toxic and nontoxic close relatives within Dinophysis, Karenia and Aza- dinium. In metabarcoding studies, sequences are clustered at a given ORCID similarity level (98% or 97%) to avoid inflating diversity estimates (Massana et al., 2015; Onda et al., 2017; Smith et al., 2017; de Vargas Diana Sarno http://orcid.org/0000-0001-9697-5301 et al., 2015). 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